A series of subclones were isolated from the cloned but phenotypically heterogeneous MC3T3-E1 cell line. The subclones were selected for high or low osteoblast differentiation and mineralization after growth in medium containing ascorbic acid. The MC3T3-E1 Subclone 4 (ATCC CRL-2593) and the MC3T3 Subclone 14 (ATCC CRL-2594) lines exhibit high levels of osteoblast differentiation after growth in ascorbic acid and 3 to 4 mM inorganic phosphate. [51540] They form a well mineralized extracellular matrix (ECM) after 10 days. The MC3T3 Subclone 24 (ATCC CRL-2595) and the MC3T3 Subclone 30 (ATCC CRL-2596) lines exhibit poor osteoblast differentiation after growth in ascorbic acid. They do not form ECM. They can be used as negative controls for Subclones 4 and 14. RefWang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097

The base medium for this cell line is Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine and 1 mM sodium pyruvate, but without ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001). To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Subculturing

Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.